The major objective of this proposal is to elucidate the lineage relationships between the different cell types in the developing limb and heart. Lineage relationships in the limb will be determined in the chick using a powerful technique involving injection of embryonic progenitors with a library of retroviral vectors. Each vector carries a distinct "tag" and subsequent PCR sequencing of the tag from injected cells is used to define individual cells in the later limb bud or mature limb that are clonally related. Critical questions to be addressed by this analysis in the limb include the timing of specification and lineage relationships of various cell types within the limb bud, whether there are distinct dorsal and ventral compartments in the limb bud, and the time when proximodistal limb segments are specified within the limb bud. We will also conduct an analysis of the lineages contributing to various portions of the valves of the heart. Heart valves are currently thought to derive from delanination of the endocardium, forming a precursor population called the cardiac cushion. However, we have indications that the cushions may form from 3 distinct cell populations, each with different fate in the mature valve. This will be tested using recombinase-based fate mapping in mouse embryos. In addition to addressing specific questions in limb and heart development, these studies will provide an important context for designing future experiments. Finally, the reagents generated in these studies will also allow us to definitely test whether the tendon and cartilage precursors in the limb bud are dedicated progenitors or whether they contribute to other cell types in the limb. Moreover, the same reagents will be used to test whether Hox gene expression is required autonomously in the tendon and skeletal progenitors for proper patterning of the limb.